This invention relates to a method of preparing biological material obtained from a biological sample, more particularly a blood or sputum sample.
Methods for preparing biological material obtained from blood or sputum samples have been known for decades. These methods are based on multiple steps of preparing and purifying the biological material. For example, normally, the known methods for preparing and purifying nucleic acids include the steps of:                separating of white cell fraction from the balance of the blood sample through centrifugation;        lysing the white cell fraction with a detergent;        digesting the white cell fraction with proteinase;        extracting the biological material from the digested white cell fraction with organic solvents such as phenol; and        precipitating the biological material by adding alcohol.        
The precipitated biological material is subsequently analysed or stored or transported for later analysis.
A first disadvantage of these methods is thus that, owing to the multiple steps, the methods are laborious and time consuming. Another disadvantage experienced with this method is that the addition of enzymes and reagents in the form of detergents and solvents interferes with the analysis of the biological material. Yet another disadvantage of these methods is that owing to the relative complexity of the steps, and the nature of the reagents used, the implementation of these methods are confined mostly to laboratories and are not suitable to be exercised in the field where blood samples are collected.
A further disadvantage of the known methods of preparing biological material from blood samples is that they require regular manual handling of the samples, thereby increasing risk for laboratory personnel to be infected with hepatitis virus, human immunodeficiency virus (HIV) as well as other pathogens present in the blood samples. Also, it was found that with the known methods, there is an increased risk for cross-sample contamination, potentially leading to false positive results. This could have devastating effects in cases where a person is incorrectly diagnosed with HIV.
Both Boom et al. (1989) and Bush et al. (1991) disclose a method for preparing biological material, such as DNA, by using a strong chaotropic agent, guanidinium thiocyanate (GuSCN), for lysing human cells, and followed by sorbtion of DNA to glass powder. These methods use the ability of glass-based sorbents to bind DNA or nucleic acids at high salt concentrations and release at low salt concentrations.
A disadvantage of the method proposed by Boom et al. (1989) and Bush et al. (1991) is that it consists of multiple steps, including lysing, binding and several washing steps with different buffers and is therefore not suitable for the rapid or immediate preparation of biological material for analysis from the blood samples. In particular, the method is not suitable for preparing nucleic acid material from whole blood samples, owing to the presence of a large amount of proteins in the blood. In addition, without the inclusion of several washing steps, the nucleic acids are contaminated by red blood cells, thereby inhibiting PCR amplification.
Furthermore, various methods of capturing biological material from the biological samples, such as blood and tissue, have been developed and are commercially available. For example, U.S. Pat. No. 5,346,994 discloses a method of isolating substantially pure RNA, DNA and proteins from biological tissue, comprising the steps of:                (a) homogenising a tissue sample in a solvent solution comprising effective amounts of phenol, a guanidinium compound and a thiocyanate compound selected from the group consisting of ammonium thiocyanate and sodium thiocyanate for extracting substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from biological tissue to form a homogenate;        (b) adding a water-insoluble organic solvent to said homogenate and sedimenting it to form a mixture consisting of an aqueous phase containing substantially pure, undegraded RNA, an organic phase containing proteins, and an interphase containing substantially pure, undegraded DNA;        (c) precipitating RNA from the aqueous phase by the addition of a lower alcohol thereto and recovering the precipitated RNA by sedimentation;        (d) extracting the organic phase and interphase with water;        (e) precipitating proteins from the organic phase by the addition of a lower alcohol thereto and recovering the precipitated proteins by sedimentation; and        (f) precipitating DNA from the interphase by the addition of CsCl, sodium citrate solution and a lower alcohol thereto and recovering the precipitated DNA by sedimentation.        
From the above it is clear that the method of the above patent is not only time consuming and relatively complex, but also limited to the use of specific enzymes and reagents.
There is therefore clearly a long-standing need for an efficient and robust method for preparing uncontaminated biological material from a blood sample without applying numerous consecutive steps that necessarily have to be taken in a laboratory environment.
US patent application number US2010/0291536A1 (“the '563 application”) discloses a method and device for collecting, treating and analysis of biological material by introducing a source material into a specimen container, transferring the source material to a processing device and thermally, chemically and/or mechanically treating the source material to alter at least one constitutive characteristic of the source material and to release or create a target material from the source material. Furthermore, paragraph 23 on page 3 of the specification states that the source material may include blood and sputum, amongst other. However, a disadvantage experienced with the method disclosed in the '563 application, as confirmed by the inventors thereof, was that they were unable to successfully apply the method disclosed in the specification to blood samples.
A further disadvantage of the invention disclosed in the '563 application is that it requires two distinct steps taking place in two separate chambers or wells. The inventors of the present invention thus embarked on research aimed at improving the method disclosed in the '563 application to the extent that it could be successfully applied to blood samples using steps occurring simultaneously in a single chamber.